CHROMO-LAL - Associates of Cape Cod, Inc.

1 limulus amebocyte lysate REAGENTS does not reduce the level of endotoxin detectable in the limulus amebocyte lysate CHROMO-LAL . Reagents required to perform the CHROMO-LAL Assay are listed below. Unopened reagents are stable at 2-8 C until sample (endotoxin may adsorb to the container surface). Many substances interfere with the CHROMO-LAL test. for the Detection and Quantitation of Gram Negative the expiration date printed on the container label. Before Interference is indicated by inhibition, the recovery of less CHROMO-LAL Bacterial Endotoxins (Lipopolysaccharides) reconstitution, bring the reagents to room temperature endotoxin than known to be present in a sample; or by and tap the vials containing lyophilized material against a enhancement, the recovery of more endotoxin than the SUMMARY AND EXPLANATION OF TEST hard surface to cause loose material to fall to the bottom of amount known to be present (see "Procedure" for detec- Manufactured by: Telephone: (508) 540-3444.)

2 In the 1950's, Frederik Bang observed that infection from the vial. tion of interference). Interference is generally overcome by Toll-Free: (888) 395-2221. gram negative bacteria resulted in intravascular coagula- diluting the sample with LAL Reagent Water. Do not dilute Fax: (508) 540-8680 1. CHROMO-LAL , limulus amebocyte lysate co- Technical Support: (800) 848-3248 tion in limulus polyphemus, the horseshoe crab (1). Levin beyond the Maximum Valid Dilution (MVD; 10, 11, 12, 13). lyophilized with chromogenic substrate Customer Service: (800) 525-8378 and Bang demonstrated that the coagulation was caused This reagent is an aqueous extract of amebocytes of L. by the activation of a number of enzymes located in the polyphemus, buffered at pH 7, and co-lyophilized with US License # 700 PN001087 Rev2 March 2014.

3 Blood cells (amebocytes) of limulus polyphemus, and that endotoxin limit x product concentration the chromogenic substrate. Reconstitute CHROMO-LAL . this activation was initiated by the endotoxin (lipopolysac- immediately before use with mL LAL Reagent Water MVD = . charide) in the gram negative bacterial cell walls (2, 3, 4). (LRW). This solution is stable 24 hours at 2-8 C or for Subsequently, the limulus amebocyte lysate (LAL) test two weeks at -20 C or colder if frozen immediately after Certain compounds may need special treatment in addi- using LAL reagent prepared from horseshoe crab blood reconstitution and not contaminated. CHROMO-LAL . tion to dilution to remove interference. For example, was shown to be the most sensitive and specific means of may be frozen and thawed once.

4 Contamination may blood products containing activated enzymes may cause measuring bacterial endotoxins (5). The chromogenic test, be indicated by a dark yellow color that develops rapid- false positive results. These types of samples may be introduced in 1977 (6, 7), is a modification that enables ly after reconstitution. The reagent will turn yellow diluted with LAL Reagent Water and heated at a minimum endotoxin concentration to be measured as a function of slowly under normal conditions of use. temperature of 75 C for a period of time shown to elimi- color intensity rather than by turbidity or gelation in the 2. Control Standard Endotoxin (CSE) nate interference without loss of endotoxin activity. reaction mixture. Results obtained by this modified Control Standard Endotoxin (CSE) is not provided Samples that absorb strongly at 405 nm may interfere method are generally comparable to those obtained by the with CHROMO-LAL and must be ordered separately.

5 With the test and may require prior dilution. gel-clot or turbidimetric methods within the error of the CSE obtained from Associates of Cape Cod, Inc., is tests. used to construct standard curves, validate product, TEST PROCEDURE. In the CHROMO-LAL test, co-lyophilized LAL and substrate and prepare inhibition controls. Each vial contains a Reagents required to perform the CHROMO-LAL Assay are reagent are mixed with test sample in a microplate and measured weight of endotoxin. USP Endotoxin listed under reagents. incubated in a reader at 37 1 C. Absorbance measure- Reference Standard may be obtained from the ments are collected with time after addition of Chromo- Pharmacopeial Convention, Inc. Follow manufacturer's Equipment and materials required but not provided: LAL and analyzed by suitable software.

6 The time (onset directions for reconstitution and storage of standard 1. Test tubes and/or microplates free of detectable endo- time) taken for a sample to reach a specified absorbance endotoxins. CSE lots may show different potencies toxin. Both available through Associates of Cape Cod, (onset OD) is calculated; and a standard curve, showing (EU/ng) when tested with different lots of Chromo- Inc. the linear correlation between the log onset time and the LAL. If using CSE, endotoxin concentrations can be 2. Pipettes and pipette tips that are free of detectable log concentration of standard endotoxin, is generated. The expressed in EU/mL if the potency of a given lot of endotoxin. Available through Associates of Cape Cod, maximum range of endotoxin concentrations for the stan- CSE has been determined with the CHROMO-LAL lot in dard curve is EU/mL - 50 EU/mL.

7 The sensitivity ( ) question. (11,12). of the assay is defined as the lowest concentration used in 3. Repetitive pipettes with dispensing syringes free of 3. LAL Reagent Water (LRW). the standard curve. The maximum sensitivity of this test is detectable endotoxin. LRW is sterile water prepared by distillation or reverse EU/mL. osmosis that shows no detectable endotoxin when 4. Vortex mixer. tested with CHROMO-LAL . Additional vials of LRW 5. Microplate reader equipped with suitable. software and BIOLOGICAL PRINCIPLE may be obtained from Associates of Cape Cod, Inc. capable of maintaining a uniform temperature across LAL contains enzymes that are activated in a series of Precautions and Warnings: CHROMO-LAL is for in vitro the microplate of 37 1 C.

8 Available through Associates reactions in the presence of endotoxin. The last enzyme diagnostic use only. Do not use these reagents for the of Cape Cod, Inc. activated in the cascade splits the chromophore, para- nitro aniline (pNA), from the chromogenic substrate, detection of endotoxemia. Exercise caution when han- 6. Kinetic software. Software that collects and stores opti- producing a yellow color. dling CHROMO-LAL reagent because its toxicity has not cal density (OD) readings at short intervals is necessary. been determined and allergies to LAL have been reported The software must also calculate onset time for the Endotoxin (8). Correct application of this test requires strict adher- sample in each well. An onset time is the time taken 1.

9 Proenzyme Enzyme ence to all items in the recommended procedure. Aseptic for the OD in a given well to reach a specified OD value technique must be used. All materials coming in contact (onset OD). The chosen value may be between Enzyme with specimens and reagents must be free of detectable and OD units; however the same value should be 2. Chromogenic Substrate Peptide + pNA. endotoxin. Heat stable materials, including clean glass- used for routine testing as was used for the validation ware, may be rendered free of detectable endotoxin by of the assay for that product. The amount of pNA released and measured photometri- exposure to dry heat at a minimum temperature of 250 C. The software should generate the standard curve cally at 405 nm is proportional to the amount of the endo- for a minimum of 30 minutes (9).

10 Parameters (slope, intercept, and correlation coeffi- toxin in the system. The greater the endotoxin concentra- cient) and calculate the endotoxin concentrations in tion, the faster the reaction. SPECIMEN COLLECTION AND PREPARATION. the unknown samples. The software may perform Collect samples in a way that avoids microbial contamina- additional calculations such as calculating the concen- tion. Use aseptic technique when handling specimens and tration of the endotoxin recovered in the positive reagents. Test any samples as soon as possible after collec- product control after subtraction of any endogenous tion; otherwise store them at 2-8 C. If bacterial growth is endotoxin in the sample. expected, samples may be frozen. Confirm that storage.